KEYWORDS
Hybridoma, genome-scale metabolic model, antiapoptotic gene, mitochondrial transport, BHRF1
1.- INTRODUCTION
The demand for monoclonal antibodies has severally increased over the last years, mainly for the new applications in therapy, but also for clinical diagnosis and highly specific purification processes (Walsh, 2018; Grilo and Mantalaris, 2019). In this regard, the capacity of mammalian cells to perform complex post-translational modifications to yield biologically active proteins has led them to be the preferred system for biopharmaceuticals production. About 70-80% of all biopharmaceuticals, including monoclonal antibodies, viral vaccines and gene therapy vectors, are produced with mammalian cells; and among the top ten selling protein biopharmaceuticals in 2014, six of them are antibodies or antibody-derived proteins. It is therefore not surprising that monoclonal antibodies-based drugs production using mammalian cell-based systems in the 2016 reached almost the double of the 2010 value (Estes and Melville, 2013; Walsh, 2018). In the last years, monoclonal antibodies industrial manufacturing has been based on mammalian cell lines such as hybridoma among others (Wurm, 2004; Coco-Martin and Harmsen, 2008; Estes and Melville, 2013; Hnasko and Stanker, 2015).
Mammalian cell-based processes present an important culture limitation: the accumulation of metabolic by-products (i.e. lactate and ammonia) up to cytotoxic concentration, as well as the depletion of essential nutrients, triggers the apoptosis (programmed cell death) (Arden and Betenbaugh, 2004). The prevention of apoptosis during the cell growth has a critical effect on final process productivity: The increase of cell life-span results in an increase of product of interest synthesis and accumulation, since cells remain productive for a longer time, even after the exponential cell growth phase (Zhang et al., 2018). Moreover, more robust cell lines less sensible to apoptosis allow designing high cell density culture strategies based on keeping low nutrients concentration in narrower ranges (Casablancas et al., 2013).
In the last years, the strategies to generate stress-resistant cell lines preventing apoptosis have been focused on blocking the apoptotic transduction pathways (Smolke, 2010; Henry et al., 2020). Although there are different pathways controlling the activation of signaling cascades of cell apoptosis activation, many of the apoptosis signals converge on the mitochondria, which stores numerous molecules that activate apoptosis (Arden and Betenbaugh, 2004). The most used strategy to prevent apoptosis has been the overexpression of bcl-2 or bcl-xL genes, which inhibits the release of pro-apoptotic molecules from the mitochondria (Smolke, 2010; Vives et al., 2003b). This strategy has been successfully applied in different mammalian cell lines, as CHO or hybridoma, showing higher viabilities and improved robustness in cell culture (Fussenegger et al., 2000; Mastrangelo et al., 2000; Simpson et al., 1997; Tey et al., 2000). The expression of mcl-1, another antiapoptotic gene with similar mechanism to bcl-2/bcl-xL, has also shown good results (Majors et al., 2009). Another approach has been targeting directly the caspase cascade, expressing X-linked inhibitor of apoptosis (XIAP) or cytokine response modifier CrmA, which both act inhibiting directly the caspases (Sauerwald et al., 2003). The expression of different viral proteins has also been reported to have antiapoptotic effects in hybridoma cell cultures, as ksblc-2 from Karposi’s sarcoma-associated herpesvirus (Vives et al., 2003a) and bhrf-1 from Epstein-Barr virus (Juanola et al., 2009). Furthermore, the downregulation of the pro-apoptotic genes Bak, Bax and Casp3 has shown to reduce the apoptosis in CHO cells (Xiong et al., 2019).
Additionally, anti-apoptotic genes have shown an effect on metabolism, although this is not fully understood yet (Dorai et al., 2009; Templeton et al., 2014). This is remarkable as cell-based processes present an important limitation regarding the metabolism: deregulated substrates uptake (high consumption rates of mainly glucose and glutamine), what is linked to the secretion and accumulation of lactate and ammonia as by-products of the metabolism (Martínez-Monge et al., 2018a).
The reduction of the secretion and accumulation of lactate remains a hot topic for biomanufacturing industry. Many different approaches have been explored to reduce or delay lactate generation in cell culture including media design by substitution of glucose for alternative carbon sources like fructose or galactose (Altamirano et al., 2006), different fed-batch strategies limiting glucose concentration (Casablancas et al., 2013; Zhang et al., 2004) and several cell engineering approaches as the expression of pyruvate carboxylase (Henry and Durocher, 2011) and downregulation of lactate dehydrogenase (Chen et al., 2001). In all the different scenarios described, a reduction of lactate accumulation up to a certain extend was observed but never totally depleted.
In the present work, a murine hybridoma cell line (KB-26.5) has been engineered to over-express BHRF-1 protein (KB26.5-BHRF1). Besides the protective effect against apoptosis achieved, the over-expression of BHRF-1 had an unexpected direct effect on cell physiology and metabolism. Mainly higher cell growth rate and more efficient nutrient usage were observed in batch cultures, significantly reducing the lactate generation. Therefore, metabolic flux balances techniques were applied to better understand the interactions and effects of BHRF-1 protein expression with cell metabolism. The interpretation of the intracellular fluxes obtained allowed an understanding of their effects on the improved cell metabolism and also allowed to arise some hypothesis about the possible BHRF-1 interactions with the metabolic pathways. Nowadays, prevention of apoptosis remains as a relevant research topic as it has been addressed in a recent review by Henry et al., (2020), in which the effect of different anti-apoptotic strategies applied in the last years were presented and discussed.